Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth.
The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.
Wise
D J
DJ
Virginia Polytechnic Institute and State University, Department of Biochemistry, Blacksburg 24061-0308, USA.
Anderson
C D
CD
Anderson
B M
BM
D001667:Binding, Competitive; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D006190:Haemophilus / Q000187:drug effects* / Q000201:enzymology* / Q000254:growth & development; D006358:Hot Temperature; D007700:Kinetics; D009536:Niacinamide / Q000031:analogs & derivatives / Q000494:pharmacology; D011489:Protein Denaturation; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D013104:Spheroplasts / Q000201:enzymology; D013379:Substrate Specificity; D014462:Ultrafiltration
D004791:Enzyme Inhibitors; D012265:Ribonucleotides; D009536:Niacinamide; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase